The objective of this project is to develop high precision isotope ratio monitoring chromatography (IRM-C), based on mass spectrometry (MS) as a tool for biomedical research. Presently, this novel technology permits isotope ratio determinations for G C effluent at 100 x more precision than conventional GC/MS. The technique will be combined with the use of highly enriched, uniformly 13 C-labeled biomolecules produced by algae to permit tracer experiments with improved detectability compared to traditional radiotracers without the health risks to subjects associated with radionuclides. Therefore, studies previously limited to animal models will be possible directly in all humans, including vulnerable groups such as infants, children, and pregnant and lactating women. A decline in animal use should be a direct result of this development. Since the technique was developed for studies of natural variation in isotope ratio, the present state-of-the-technology still suffers certain disadvantages relative to radiotracer studies and is therefore limited in its applicability to biomedical tracer research. The specific aims of this project will overcome these limits and add important capabilities not possible in radiotracer experiments: expansion of the dynamic range over which precise isotope ratios are obtained from 2 to 5 orders of magnitude; construction of an interface to facilitate high precision IRM-liquid chromatography (LC), which will permit applications to most biomolecules; design of a system for hydrogen high precision IRM-GC/LC to permit the use of deuterium-labeled compounds and double label experi- ments; interface of the system to a full scan quadrupole mass spectrometer for direct determination of metabolite structure. Human fatty acid metabolism will serve as a specific biomedical research area to evaluate and demonstrate the use of instrumental and methodological developments. Isotopic reference materials for individual fatty acids and fatty acid methyl esters will be established, maintained, and provided to the user community. Studies of fatty acid biokinetics, interconversion, and transformation will be executed in adults as each thrust area develops. The computer program CONSAAM will be used for biokinetic modeling of fatty acid transport and transformation as components of specific lipid classes in lipoproteins and plasma borne cell types.